Dual A1 and A2A adenosine receptor antagonists, methoxy substituted 2-benzylidene-1-indanone, suppresses intestinal postprandial glucose and attenuates hyperglycaemia in fructose-streptozotocin diabetic rats.

BACKGROUND/AIM: Recent research suggests that adenosine receptors (ARs) influence many of the metabolic abnormalities associated with diabetes. A non-xanthine benzylidene indanone derivative 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1 H-inden-1-one (2-BI), has shown to exhibit higher affinity at A1/A2A ARs compared to caffeine. Due to its structural similarity to caffeine, and the established antidiabetic effects of caffeine, the current study was initiated to explore the possible antidiabetic effect of 2-BI. METHODS: The study was designed to assess the antidiabetic effects of several A1 and/or A2A AR antagonists, via intestinal glucose absorption and glucose-lowering effects in fructose-streptozotocin (STZ) induced diabetic rats. Six-week-old male Sprague-Dawley rats were induced with diabetes via fructose and streptozotocin. Rats were treated for 4 weeks with AR antagonists, metformin and pioglitazone, respectively. Non-fasting blood glucose (NFBG) was determined weekly and the oral glucose tolerance test (OGTT) was conducted at the end of the intervention period. RESULTS: Dual A1/A2A AR antagonists (caffeine and 2-BI) decreased glucose absorption in the intestinal membrane significantly (p < 0.01), while the selective A2A AR antagonist (Istradefylline), showed the highest significant (p < 0.001) reduction in intestinal glucose absorption. The selective A1 antagonist (DPCPX) had the least significant (p < 0.05) reduction in glucose absorption. Similarly, dual A1/A2A AR antagonists and selective A2A AR antagonists significantly reduced non-fast blood glucose and improved glucose tolerance in diabetic rats from the first week of the treatment. Conversely, the selective A1 AR antagonist did not reduce non-fast blood glucose significantly until the 4th week of treatment. 2-BI, caffeine and istradefylline compared well with standard antidiabetic treatments, metformin and pioglitazone, and in some cases performed even better. CONCLUSION: 2-BI exhibited good antidiabetic activity by reducing intestinal postprandial glucose absorption and improving glucose tolerance in a diabetic animal model. The dual antagonism of A1/A2A ARs presents a positive synergism that could provide a new possibility for the treatment of diabetes.

Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A1 Antagonists.

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A1AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R4 and R6 of the pyrimidine core but most importantly the prominent role to the unprecedented A1AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A1 and A2AARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

Chalcone-inspired rA1 /A2A adenosine receptor ligands: Ring closure as an alternative to a reactive substructure.

Over the past few years, great progress has been made in the development of high-affinity adenosine A1 and/or A2A receptor antagonists-promising agents for the potential treatment of Parkinson's disease. Unfortunately, many of these compounds raise structure-related concerns. The present study investigated the effect of ring closures on the rA1 /A2A affinity of compounds containing a highly reactive α,ß-unsaturated carbonyl system, hence providing insight into the potential of heterocycles to address these concerns. A total of 12 heterocyclic compounds were synthesised and evaluated in silico and in vitro. The test compounds performed well upon qualitative assessment of drug-likeness and were generally found to be free from potentially problematic fragments. Most also showed low/weak cytotoxicity. Results from radioligand binding experiments confirm that heterocycles (particularly 2-substituted 3-cyanopyridines) can replace the promiscuous α,ß-unsaturated ketone functional group without compromising A1 /A2A affinity. Structure-activity relationships highlighted the importance of hydrogen bonds in binding to the receptors of interest. Compounds 3c (rA1 Ki  = 16 nM; rA2A Ki  = 65 nM) and 8a (rA1 Ki  = 102 nM; rA2A Ki  = 37 nM), which both act as A1 antagonists, showed significant dual A1 /A2A affinity and may, therefore, inspire further investigation into heterocycles as potentially safe and potent adenosine receptor antagonists.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.

Adenosine Receptor Ligands: Coumarin-Chalcone Hybrids as Modulating Agents on the Activity of hARs.

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin-chalcone hybrids were synthesized (compounds 1-8) and screened in radioligand binding (hA1, hA2A and hA3) and adenylyl cyclase (hA2B) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin-chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA1 or hA3 subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA1, while the methoxy counterparts were selective for the hA3. The most potent hA1 ligand was compound 7 (Ki = 17.7 µM), whereas compound 4 was the most potent ligand for hA3 (Ki = 2.49 µM). In addition, docking studies with hA1 and hA3 homology models were established to analyze the structure-function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson's Disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.

Design and pharmacological profile of a novel covalent partial agonist for the adenosine A1 receptor.

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746's apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746's irreversible activation of an A1 receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.

New Insights into Key Determinants for Adenosine 1 Receptor Antagonists Selectivity Using Supervised Molecular Dynamics Simulations.

Adenosine receptors (ARs), like many otherGprotein-coupledreceptors (GPCRs), are targets of primary interest indrug design. However, one of the main limits for the development of drugs for this class of GPCRs is the complex selectivity profile usually displayed by ligands. Numerous efforts have been madefor clarifying the selectivity of ARs, leading to the development of many ligand-based models. The structure of the AR subtype A1 (A1AR) has been recently solved,providing important structural insights. In the present work, we rationalized the selectivity profile of two selective A1AR and A2AAR antagonists, investigating their recognition trajectories obtained by Supervised Molecular Dynamics from an unbound state and monitoring the role of the water molecules in the binding site.

A Taxicab geometry quantification system to evaluate the performance of in silico methods: a case study on adenosine receptors ligands.

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

The Signaling Pathways Involved in the Anticonvulsive Effects of the Adenosine A1 Receptor.

Adenosine acts as an endogenous anticonvulsant and seizure terminator in the brain. Many of its anticonvulsive effects are mediated through the activation of the adenosine A1 receptor, a G protein-coupled receptor with a wide array of targets. Activating A1 receptors is an effective approach to suppress seizures. This review gives an overview of the neuronal targets of the adenosine A1 receptor focusing in particular on signaling pathways resulting in neuronal inhibition. These include direct interactions of G protein subunits, the adenyl cyclase pathway and the phospholipase C pathway, which all mediate neuronal hyperpolarization and suppression of synaptic transmission. Additionally, the contribution of the guanyl cyclase and mitogen-activated protein kinase cascades to the seizure-suppressing effects of A1 receptor activation are discussed. This review ends with the cautionary note that chronic activation of the A1 receptor might have detrimental effects, which will need to be avoided when pursuing A1 receptor-based epilepsy therapies.

Accuracy and Precision of the Receptorial Responsiveness Method (RRM) in the Quantification of A1 Adenosine Receptor Agonists.

The receptorial responsiveness method (RRM) is a procedure that is based on a simple nonlinear regression while using a model with two variables (X, Y) and (at least) one parameter to be determined (cx). The model of RRM describes the co-action of two agonists that consume the same response capacity (due to the use of the same postreceptorial signaling in a biological system). While using RRM, uniquely, an acute increase in the concentration of an agonist (near the receptors) can be quantified (as cx), via evaluating E/c curves that were constructed with the same or another agonist in the same system. As this measurement is sensitive to the implementation of the curve fitting, the goal of the present study was to test RRM by combining different ways and setting options, namely: individual vs. global fitting, ordinary vs. robust fitting, and three weighting options (no weighting vs. weighting by 1/Y2 vs. weighting by 1/SD2). During the testing, RRM was used to estimate the known concentrations of stable synthetic A1 adenosine receptor agonists in isolated, paced guinea pig left atria. The estimates were then compared to the known agonist concentrations (to assess the accuracy of RRM); furthermore, the 95% confidence limits of the best-fit values were also considered (to evaluate the precision of RRM). It was found that, although the global fitting offered the most convenient way to perform RRM, the best estimates were provided by the individual fitting without any weighting, almost irrespective of the fact whether ordinary or robust fitting was chosen.

The effect of dimerization on the activation and conformational dynamics of adenosine A1 receptor.

The adenosine A1 receptor (A1R) is one of four adenosine receptors in humans, which are involved in the function of the cardiovascular, respiratory and central nervous systems. Experimental results indicate that A1R can form a homodimer and that the protomer-protomer interaction in the A1R dimer is related to certain pharmacological characteristics of A1R activation. In this work, we performed docking, metadynamics simulation, conventional molecular dynamics simulations, Gaussian-accelerated molecular dynamics simulations, potential of mean force calculations, dynamic cross-correlation motions analysis and community network analysis to study the binding mode of 5'-N-ethylcarboxamidoadenosine (NECA) to A1R and the effect of dimerization on the activation of A1R. Our results show that NECA binds to A1R in a similar mode to adenosine in the A1R crystal structure and NECA in the A2AR crystal structure. The A1R homodimer can be activated by one or two agonists with NECA occupying its orthosteric pockets in one (which we call the NECA-A1R system) or both protomers (which we call the dNECA-A1R system). In the NECA-A1R system, activation is predicated in the protomer without NECA bound. In the dNECA-A1R system, only one protomer achieves the active state. These findings suggest an asymmetrical activation mechanism of the homodimer and a negative cooperativity between the two protomers. We envision that our results may further facilitate the drug development of A1R.

An Advanced In Silico Modelling of the Interaction between FSCPX, an Irreversible A1 Adenosine Receptor Antagonist, and NBTI, a Nucleoside Transport Inhibitor, in the Guinea Pig Atrium.

In earlier studies, we generated concentration-response (E/c) curves with CPA (N6-cyclopentyladenosine; a selective A1 adenosine receptor agonist) or adenosine, in the presence or absence of S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine (NBTI, a selective nucleoside transport inhibitor), and with or without a pretreatment with 8-cyclopentyl-N3-[3-(4-(fluorosulfonyl)-benzoyloxy)propyl]-N1-propylxanthine (FSCPX, a chemical known as a selective, irreversible A1 adenosine receptor antagonist), in isolated, paced guinea pig left atria. Meanwhile, we observed a paradoxical phenomenon, i.e. the co-treatment with FSCPX and NBTI appeared to enhance the direct negative inotropic response to adenosine. In the present in silico study, we aimed to reproduce eight of these E/c curves. Four models (and two additional variants of the last model) were constructed, each one representing a set of assumptions, in order to find the model exhibiting the best fit to the ex vivo data, and to gain insight into the paradoxical phenomenon in question. We have obtained in silico evidence for an interference between effects of FSCPX and NBTI upon our ex vivo experimental setting. Regarding the mechanism of this interference, in silico evidence has been gained for the assumption that FSCPX inhibits the effect of NBTI on the level of endogenous (but not exogenous) adenosine. As an explanation, it may be hypothesized that FSCPX inhibits an enzyme participating in the interstitial adenosine formation. In addition, our results suggest that NBTI does not stop the inward adenosine flux in the guinea pig atrium completely.

The heterotetrameric structure of the adenosine A1-dopamine D1 receptor complex: Pharmacological implication for restless legs syndrome.

Dopaminergic and purinergic signaling play a pivotal role in neurological diseases associated with motor symptoms, including Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, Huntington disease, Restless Legs Syndrome (RLS), spinal cord injury (SCI), and ataxias. Extracellular dopamine and adenosine exert their functions interacting with specific dopamine (DR) or adenosine (AR) receptors, respectively, expressed on the surface of target cells. These receptors are members of the family A of G protein-coupled receptors (GPCRs), which is the largest protein superfamily in mammalian genomes. GPCRs are target of about 40% of all current marketed drugs, highlighting their importance in clinical medicine. The striatum receives the densest dopamine innervations and contains the highest density of dopamine receptors. The modulatory role of adenosine on dopaminergic transmission depends largely on the existence of antagonistic interactions mediated by specific subtypes of DRs and ARs, the so-called A2AR-D2R and A1R-D1R interactions. Due to the dopamine/adenosine antagonism in the CNS, it was proposed that ARs and DRs could form heteromers in the neuronal cell surface. Therefore, adenosine can affect dopaminergic signaling through receptor-receptor interactions and by modulations in their shared intracellular pathways in the striatum and spinal cord. In this work we describe the allosteric modulations between GPCR protomers, focusing in those of adenosine and dopamine within the A1R-D1R heteromeric complex, which is involved in RLS. We also propose that the knowledge about the intricate allosteric interactions within the A1R-D1R heterotetramer, may facilitate the treatment of motor alterations, not only when the dopamine pathway is hyperactivated (RLS, chorea, etc.) but also when motor function is decreased (SCI, aging, PD, etc.).

Probe dependence of allosteric enhancers on the binding affinity of adenosine A1 -receptor agonists at rat and human A1 -receptors measured using NanoBRET.

BACKGROUND AND PURPOSE: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A1 -receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A1 -receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A1 -receptor agonists. EXPERIMENTAL APPROACH: A Nano-luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N-terminal Nluc-tagged A1 -receptors expressed in HEK293T cells in conjunction with both fluorescent A1 -receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645. KEY RESULTS: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A1 -receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A1 -receptor agonists in a species-dependent manner that involved increases in Bmax and pKD . CONCLUSIONS AND IMPLICATIONS: These results demonstrate the power of the NanoBRET ligand-binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A1 -receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A1 -receptors to an active agonist conformation (R*).

2-Benzylidene-1-Indanone Analogues as Dual Adenosine A1/A2a Receptor Antagonists for the Potential Treatment of Neurological Conditions.

Previous studies explored 2-benzylidine-1-tetralone derivatives as innovative adenosine A1 and A2A receptor antagonists for alternative non-dopaminergic treatment of Parkinson's disease. This study's aim is to investigate structurally related 2-benzylidene-1-indanones with substitutions on ring A and B as novel, potent and selective adenosine A1 and A2A receptor blockers. 2-Benzylidene-1-indanone derivatives were synthesised via acid catalysed aldol condensation reactions and evaluated via radioligand binding assays to ascertain structure activity relationships to govern A1 and A2A AR affinity. The results indicated that hydroxy substitution at C4 of ring A and meta (3'), or para (4') substitution on ring B of the 2-benzylidene-1-indanone scaffold (2C: ) is preferred over substitution at C5 (2D: ) or C6 (2E: ) of ring A for adenosine A1 receptor activity and selectivity in the micromolar range. Furthermore, substitution at the meta (3') position of ring B with chlorine lead to the highly potent and selective adenosine A2A receptor antagonist, compound 2 H: . Compound 2C: and the 2Q: behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. In view of these findings, compounds 2C: , 2 H: , 2Q: and 2P: are potent and selective adenosine A1 and A2A receptor antagonists for the potential treatment of neurological conditions.

Structural Basis for Binding of Allosteric Drug Leads in the Adenosine A1 Receptor.

Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A1 receptor (A1AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A1AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A1AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A1AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A1AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A1AR, which will greatly facilitate the receptor structure-based drug design.

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes.

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A1 and A2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.